Vulnerable to a wide variety of metabolic, circulatory, toxic, microbial, and neoplastic insults, the liver is one of the most frequently injured organs in the body. In some instances, the disease is primarily localized in liver cells. For example, primary liver diseases include hereditary disorders such as Gilbert""s Syndrome, Crigler-Najjar Syndrome (either Type I or Type II), Dubin Johnson Syndrome, familial hypercholesterolemia, ornithine transcarbamoylase (OTC) deficiency, hereditary emphysema and hemophilia; viral infections such as hepatitis A, B, and non-A, non-B hepatitis; and hepatic malignancies such as hepatocellular carcinoma. Robbins, S. L. et al. (1984) Pathologic Basis of Disease (W.B. Saunders Company, Philadelphia) pp. 884-942. More often, the hepatic involvement is secondary, often to some of the most common diseases of man, such as cardiac decompensation, disseminated cancer, alcoholism, and extrahepatic infections. Robbins, S. L. et al. (1984) Pathologic Basis of Disease (W.B. Saunders Company, Philadelphia) pp. 884-942.
One of the more devastating of the above-listed liver diseases is familial hypercholesterolemia (FH). FH is a consequence of a mutation in the low density lipoprotein (LDL) receptor locus. Brown, M. S. et al. (1986) Science 232:34-47. The LDL receptor is a specific cell membrane receptor involved in the transport and metabolism of cholesterol. FH heterozygotes with one mutant allele (representing about one in 500 individuals) have, from birth, a two- to threefold elevation of plasma cholesterol leading to premature atherosclerosis and coronary heart disease in adult life. Grundy, S. M. et al. JAMA 269:3015-3023. Myocardial infarctions begin to occur in heterozygous men in the third decade and peak in the fourth and fifth decades. By the age of sixty, approximately eighty-five percent of men have experienced a myocardial infarction. Women also experience an increased incidence of myocardial infarction, but the mean age of onset is ten years later.
FH homozygotes have two mutant alleles at the LDL receptor locus and are much more severely affected. These FH homozygotes have five- to sixfold elevations in plasma cholesterol levels. These individuals develop coronary, cerebral, and peripheral vascular atherosclerosis at an early age. FH homozygotes have marked elevations of LDL in the plasma from birth. Total cholesterol levels in homozygous FH are typically greater than 500 mg/dl (normal is 200 mg/dl), and the patients frequently have decreased high density lipoprotein levels. FH homozygotes typically succumb to complications from coronary heart disease prior to age 20, with males developing the disease earlier than females. Robbins, S. L. et al. (1984) Pathologic Basis of Disease (W.B. Saunders Company, Philadelphia) pp.139-140.
While drug therapy is available for FH heterozygotes, normal levels of plasma cholesterol are difficult to achieve. In the case of the FH homozygotes, the condition cannot be treated by conventional drug therapy, and the therapeutic recourses are limited to chronic plasmapheresis or orthotopic liver transplantation.
Whole liver transplantation, which is the current therapy for a variety of liver diseases, has been employed to successfully reconstitute LDL receptors in individuals with FH, thereby lowering serum cholesterol to normal levels. Whole liver transplantation, however, is limited by the scarcity of suitable donor organs. Li, Q. et al. (1993) Human Gene Therapy 4:403-409; Kay, M. A. et al. (1992) Proc. Natl. Acad. Sci. 89:89-93. In addition to the difficulty in obtaining donor organs, the expense of liver transplantation, estimated at approximately $200,000 to $300,000 per procedure, prohibits its widespread application. Another unsolved problem is graft rejection. Foreign livers and liver cells are poorly tolerated by the recipient and are rapidly destroyed by the immune system in the absence of immunosuppressive drugs. Li, Q. et al. (1993) Human Gene Therapy 4:403-409; Bumgardner, G. L. et al. (1992) Transplantation 53:857-862. While immunosuppressive drugs may be used to prevent rejection, they also block desirable immune responses such as those against bacterial and viral infections, thereby placing the recipient at risk of infection. There is a clear need, therefore, to address the limitations of the current liver transplantation therapy as treatment for the vast array of liver disorders.
To overcome the current limitations of whole liver transplantation to treat liver disorders, the present invention provides hepatocytes, compositions including the hepatocytes, and methods for treating disorders characterized by insufficient liver function by administering the hepatocytes to subjects with such disorders. The hepatocytes of the present invention offer several advantages over whole liver transplantation to treat liver disorders. For example, the hepatocytes of the present invention are isolated from pigs, which provide a convenient, relatively inexpensive, and abundant source of hepatocytes. Moreover, in some instances, the hepatocytes of the present invention are modified such that rejection of the hepatocytes upon introduction into a xenogeneic recipient is inhibited, thereby eliminating the requirement for generalized suppression of the immune system.
Accordingly, the present invention pertains to an isolated porcine hepatocyte or an isolated population of porcine hepatocytes suitable for transplantation into a xenogeneic subject, particularly a human subject. In a preferred embodiment, the xenogeneic subject has a disorder characterized by insufficient liver function. Examples of such disorders include hereditary disorders such as Gilbert""s Syndrome, Crigler-Najjar Syndrome (either Type I or Type II), Dubin Johnson Syndrome, familial hypercholesterolemia, ornithine transcarbamoylase (OTC) deficiency, hereditary emphysema, and hemophilia; viral hepatitis, such as hepatitis A, B, and non-A, non-B hepatitis, hepatocellular carcinoma, acute liver failure, and chronic liver failure. The porcine hepatocyte(s), in unmodified form, has at least one antigen on the cell surface which is capable of stimulating an immune response against the cell in a xenogeneic subject, for example, a human. The antigen on the surface of the porcine hepatocyte is altered to inhibit rejection of the cell when introduced into a xenogeneic subject. In one embodiment, the cell surface antigen which is altered is an MHC class I antigen. This MHC class I antigen can be contacted, prior to transplantation into a xenogeneic subject, with at least one anti-MHC class I antibody, or a fragment or derivative thereof, which binds to the MHC class I antigen on the cell surface but does not activate complement or induce lysis of the cell. One example of an anti-MHC class I antibody is an anti-MHC class I F(abxe2x80x2)2 fragment, such as an anti-MHC class I F(abxe2x80x2)2 fragment of a monoclonal antibody PT85. The present invention also pertains to compositions which include porcine hepatocytes and antibodies, antibody fragments, or derivatives, which bind an antigen on the surface of the porcine hepatocytes. These compositions can be inserted into a delivery device, e.g., a syringe, e.g., a syringe pump, which facilitates the introduction of the cells into a subject. In addition, the porcine hepatocytes of the invention can be grown as a cell culture in a medium suitable to support the growth of the cells.
Porcine hepatocytes obtained from both embryonic (i.e., fetal), newborn, and adult pigs are suitable for transplantation into a xenogeneic subject. Typically, embryonic porcine hepatocytes are isolated during selected stages of gestational development. For example, hepatocytes can be isolated from an embryonic pig at a stage of embryonic development when the cells can be recognized as hepatocytes. In one embodiment, the hepatocytes are isolated between about day twenty (20) to about day twenty-five (25) of gestation and birth of the pig. In other preferred embodiments, the hepatocytes are isolated between about day thirty (30) to about day thirty-five (35) of gestation and birth of the pig, more preferably between about day twenty-five (25) and about day ninety (90) of gestation, still more preferably between about day thirty (30) and about day eighty (80) of gestation, yet more preferably between about day thirty-five (35) and about day seventy (70) of gestation, still further preferably between about day thirty-five (35) and about day fifty (50) to about day sixty (60) of gestation, and most preferably between about day thirty-five (35) and about day forty (40) of gestation.
The invention further pertains to an isolated porcine hepatocyte or an isolated population of hepatocytes isolated from a pig which is essentially free from organisms which are capable of transmitting infection or disease to a xenogeneic recipient, e.g., a human, of the cells. Categories of pathogens from which the pig are free can include parasites, bacteria, mycoplasma, and viruses. In one embodiment, the pig from which the hepatocytes are isolated is free of the following organisms: Toxoplasma, eperythrozoon, brucella, listeria, mycobacterium TB, leptospirillum, haemophilus suis, M. Hyopneumonia, porcine respiratory reproductive syndrome, rabies, pseudorabies, parvovirus, encephalomyocarditus virus, swine vesicular disease, techen (Porcine polio virus), hemagglutinating encephalomyocarditus, suipoxvirus, swine influenza type A, adenovirus, transmissible gastroenteritis virus, bovine viral diarrhea, and vesicular stomatitis virus. The cells obtained from pathogen-free pigs can be modified as described herein to inhibit rejection of the cell upon introduction into a xenogeneic subject. Preferred hepatocyte ages are described herein. The present invention also pertains to compositions which include porcine hepatocytes obtained from pathogen-free pigs and antibodies, antibody fragments, or derivatives, which bind an antigen on the surface of the porcine hepatocytes. These compositions can also be inserted into a delivery device, e.g., a syringe, e.g., a syringe pump, which facilitates the introduction of the cells into a subject.
Another aspect of the invention pertains to methods for treating disorders characterized by insufficient liver function, e.g., hereditary disorders such as Gilbert""s Syndrome, Crigler-Najjar Syndrome (either Type I or Type II), Dubin Johnson Syndrome, familial hypercholesterolemia, OTC deficiency, hereditary emphysema and hemophilia; viral infections such as hepatitis A, B, and non-A, non-B hepatitis; hepatic malignancies such as hepatocellular carcinoma, acute liver failure, and chronic liver failure, in a subject, particularly a human subject. These methods include administering to a subject having such a disorder, an isolated population of porcine hepatocytes. In one embodiment, the porcine hepatocytes which can be administered to a subject having a liver disorder are porcine hepatocytes which, in unmodified form, have at least one antigen on the cell surface which is capable of stimulating an immune response against the cell in a xenogeneic subject, for example, a human. The antigen on the surface of the porcine hepatocyte is altered to inhibit rejection of the cell when introduced into a xenogeneic subject. Examples of hepatocyte cell surface antigens and methods of altering such antigens are described herein. Preferred hepatocyte ages are also described herein. In another embodiment, the porcine hepatocytes which can be administered to a subject having a disorder characterized by insufficient liver function are porcine hepatocytes which are obtained from a pig which is essentially free from organisms which are capable of transmitting infection or disease to a xenogeneic recipient, e.g., a human, of the cells. Pathogen-free pigs are described in detail herein. In one embodiment, administration of the porcine hepatocytes is accompanied by a step wherein the portal blood pressure is decreased, e.g., using an transjugular intra-hepatic porto-systemic shunt (TIPS). Transplantation of the porcine hepatocytes can be also accompanied by administration of an immunosuppressive agent, e.g., cyclosporine A, FK506, RS-61443, or a T cell antibody, to the subject.